Preparations of vitamin D analogs, spirostanols and furostanols from diosgenin and their cytotoxic activities

Article abstract of DOI:10.1016/j.ejmech.2005.02.003

Vitamin D analogs 12 and 13 having a spiro ring in the side chain, various spirostanols 18-21, 26, 27, 29 and 37, and furostanols 34-36 having SCN and SeCN groups at the 26 position were prepared from diosgenin 1 via (20S,22R,25R)-spirost-1α,2α-epoxy-4,6-

Full text of DOI:10.1016/j.ejmech.2005.02.003

European Journal of Medicinal Chemistry 40 (2005) 662–673
www.elsevier.com/locate/ejmech
Preparations of vitamin D analogs, spirostanols and furostanols
from diosgenin and their cytotoxic activities
Hong-Ji Quan b, Jyunichi Koyanagi a, Fusao Komada a, Setsuo Saito a,*
a Faculty of Pharmaceutical Sciences, Josai University, Keyakidai 1-1, Sakado, Saitama 350-0295, Japan
b College of Pharmacy, Yanbian University, Yanji, Jilin 133000, China
Received 29 October 2004; revised and accepted 3 February 2005
Available online 07 April 2005
Abstract
Vitamin D analogs 12 and 13 having a spiro ring in the side chain, various spirostanols 18–21, 26, 27, 29 and 37, and furostanols 34–36
having SCN and SeCN groups at the 26 position were prepared from diosgenin 1 via (20S,22R,25R)-spirost-1a,2a-epoxy-4,6-dien-3-one 19
as a key intermediate. The cytotoxic activities of these derivatives as well as 1 on scarcely P-gp-expressed HCT 116 cells and P-gp-
overexpressed Hep G2 cells were examined by MTT assay. Furostanols 34 (IC50 value: 4.9 0.3 µM) and 36 (IC50 value: 1.3 0.2 µM)
exhibited marked cytotoxic effects on HCT 116 cells, and spirostanol 29 (IC50 value: 2.4 0.8 µM) and furostanol 36 (IC50 value: 2.8 0.4
µM) on Hep G2 cells. Furthermore, the effects of vitamin D analog 12, spirostanol 26 and furostanol 36 on apoptosis-signaling pathways were
investigated. Compounds 12 and 26 overexpressed p53 and Bax mRNAs, while compound 36 overexpressed only Bax mRNA.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Vitamin D analog; Spirostanol; Furostanol; Cytotoxicity; Apoptosis
1. Introduction
rhizomes of Trillium erectum Linne (Liliaceae) [7]. Com-
pound 1 has a hydroxyl group and an olefin bond at the
C-3 and C-5 positions, respectively. The E and F rings which
are a tetrahydrofuran ring and a tetrahydropyran ring, respec-
tively, of 1 were fused at the C-22 position to form a spiro
ring (Fig. 1). These functional groups, bonds and rings are
available for the structural conversion of 1, and some of the
resulting products will be useful as pharmacologically active
compounds or precursors for their synthesis (Fig. 1).
Previously [8] we reported the syntheses of diosgenin
derivatives, (20S,22S,25R)-22-thiospirost-5-en-3b-ol 2,
(20S,22S,25R)-22-selenospirost-5-en-3b-ol 3 and solasodine
4, having hetero atoms such as sulfur (S), selenium (Se) and
The overexpression of the P-glycoprotein (P-gp) which
causes multidrug resistance (MDR 1) to some cytotoxic com-
pounds in tumor cells has been a significant obstacle for suc-
cessful chemotherapy of many cancers [1–3]. Recently, a
number of modulators have been studied in detail to over-
come these obstacles [4,5]. These modulators included cal-
cium channel blockers, calmodulin antagonists, steroidal
agents, protein kinase C inhibitors, immunosuppressive drugs
and antibiotics as well as surfactants [5]. However, these
agents often give disappointing results in vivo because their
low binding affinities require the use of high doses, resulting
in unacceptable toxicity [4]. Today, the aim is to obtain use-
ful modulators and potent cytotoxic compounds which are
not influenced by efflux systems such as P-gp. We have
attempted to obtain compounds that are highly toxic to the
cancer cells which overexpress P-gp.
Diosgenin (1) is an aglycon of dioscin isolated from rhi-
zomes of Dioscorea cokoro Makino (Dioscoreaceae) [6] and
* Corresponding author. Tel.: +81 49 271 7667; fax: +81 49 271 7984.
E-mail address: saitoset@josai.ac.jp (S. Saito).
Fig. 1. Structure of diosgenin 1.
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.02.003

H.-J. Quan et al. / European Journal of Medicinal Chemistry 40 (2005) 662–673
663
nitrogen (as NH), respectively, in the F ring, a,b-unsaturated
derivatives, (20S,22S,25R)-22-thiospirost-4-en-3-one 5,
(20S,22S,25R)-22-selenospirost-4-en-3-one 6, solasodinone
7 and diosgenone 8 having S, Se, NH and O, respectively, in
the F ring, dienone derivatives, (20S,22R,25R)-22-spirost-1,4-
dien-3-one 9 and (20S,22S,25R)-22-thiospirost-1,4-dien-3-
one 10, having O and S, respectively, in the F ring, and
26-cyanoselenopseudodiosgenone 11 from 1. We have also
compared their pharmacological effects on the inhibition of
interferon-c production, cytotoxic activities, antiurease activi-
ties and antibacterial activities with those of 1. The present
study deals with preparations of vitamin D analogs having a
spiro ring and various spirostanols and furostanols contain-
ing several functional groups in the molecules from 1. Fur-
thermore, the cytotoxic activities of the synthesized com-
pounds are evaluated using human colorectal (HCT 116) and
human hepatoma (Hep G2) cancer cell lines, and also the
apoptosis-signaling pathways activated by three different
types of compounds, a vitamin D analog 12, a spirostanol 26,
and a furostanol 36, in Hep G2 cell lines are investigated using
p53, p21, Bax and Bcl-2 mRNAs [9].
structure in the B ring were utilized as starting materials
(Fig. 2). As the structure of the A ring of diosgenin 1 is the
same as that of 14 and 15, 1 was used as a starting material
for the synthesis of vitamin D analogs having a spiro ring at
the side chain (Scheme 1 and Fig. 2).
Liu et al. [18] reported the synthesis of (25R)-ruscogenin
(17) which is the aglycon of (25R)-ruscogenin-1-yl-b-D-
xylopyranosyl-(1 → 3)-[b-D-glucopyranosyl-(1 → 2)]-b-D-
fucopyranoside obtained from the tuber of Liriope muscari
(Liliaceae). Compound 17 was prepared from diosgenin 1 as
a starting material. In the synthesis of 17, epoxide 19 was
obtained from 1 via (20S,22R,25R)-spirost-1,4,6-trien-3-one
18. Then 19 was reacted with Li/NH3 in THF, followed by
treatment with NH4Cl, to obtain (20S,22R,25R)-spirost-1a,3b-
dihydroxy-5-ene (20) which was a key intermediate for the
synthesis of 17, together with (20S,22R,25R)-spirost-1a,3b-
dihydroxy-6-ene and unidentified products.As compound 20
was also thought to be an intermediate of vitamin D analog
12, which was obtained by the minor modification of the pro-
cedure reported by Liu et al. [18]; Birch reduction of 19 with
Na in liquid NH3 instead of Li in liquid NH3 gave two prod-
ucts, 20 and its epimer 21 with respect to the three position in
yields of 44.1% and 5.9%, respectively. The 1H-NMR spec-
trum of 20 was consistent with that reported by Liu et al.
Compound 21 showed the same quasimolecular ion peak as
that of 20 at m/z 431 [M + H]+ in the fast atom bombardment
mass spectrum (FABMS). The 13C-NMR spectrum of 21 was
superimposable with that of 20 (Table 1). The configuration
at the three position was confirmed by comparison of the
1H-NMR spectrum of 21 with that of 20; while the signal of
H-3a of 20 was observed as a multiplet at d 3.98, the signal
of H-3b of 21 was exhibited as a broad singlet at d 4.14. These
2. Chemical results and discussion
Our initial purpose was to obtain vitamin D type deriva-
tives 12 and 13 having a spiro ring at the side chain from 1
and to evaluate the cytotoxic activities of the derivatives
(Scheme 1). Many papers devoted to the synthesis of vitamin
D analogs have been published [10–17]. In these syntheses,
cholesterol 14 and 24-oxocholesterol 15 having a double bond
at the five position and provitamin D 16 having a cis diene
Scheme 1
. Preparations of vitamin D analogs 12 and 13. Reagents and conditions: (a) i) Na/NH3, THF, -60 °C, ii) NH4Cl; (b) Ac2O, pyridine; (c) i) NBS,
n-hexane, reflux, 2 h, ii) c-collidine, xylene, reflux 2 h; (d) KOH, MeOH, benzene, 65 °C, 1 h; (e) i) Irradiation, Et2O, 0 °C, 2 h, ii) Et2OH, reflux, 2 h.

664
H.-J. Quan et al. / European Journal of Medicinal Chemistry 40 (2005) 662–673
Fig. 2. Structures of compounds 14–17.
spectral data suggest that 21 is (20S,22R,25R)-spirost-1a,3a-
dihydroxy-5-ene.
as that of 24. The 13C-NMR spectrum of 28 was superimpos-
able with that of 24 (see Section 5.1.1.4.). The successive
irradiation and isomerization of 29 obtained from 28 gave
another vitamin D derivative 13 in 20.0% yield, which showed
the same ion peak at m/z 429 [M + H]+ as 12 in the FABMS.
Also the 13C-NMR spectrum of 13 was superimposable with
that of 12.
As we succeeded in obtaining vitamin D type derivatives
12 and 13 having a spiro ring at the side chain, we next
attempted to synthesize their analogs, such as (20S,22S,25R)-
9,10-secothiospirosta-5,7,10(19)-trien-1a,3b-diol 40 and
(20S,22S,25R)-9,10-secoselenospirosta-5,7,10(19)-trien-
1a,3b-diol 41, having a sulfur or a selenium atom instead of
an oxygen atom in the heterospiro ring. Compound 24 was
heated in acetic anhydride to give triacetate 30 in 78.3% yield,
which showed an ion peak [M + H]+ at m/z 555. The triac-
etate was hydrolyzed with methanolic KOH at room tempera-
ture to give crude triol 31 which showed an ion peak [M + H]+
at m/z 429 (Fig. 3).
With respect to p-toluenesulfonylation (tosylation) of an
alcohol,Yoshida et al. [20] recently reported that i) tosylation
of some alcohols with p-toluenesulfonyl chloride (TsCl) using
pyridine as a catalyst and solvent gave undesirable products,
ii) tosylation using a catalyst of Et3N/Me3N·HCl (ca. 20:1) in
CH2Cl2 proceeded faster than the case of i) and gave the
desired tosylates. Here we performed both tosylations. The
triol 31 reacted with TsCl using Et3N/Me3N·HCl (ca. 20:1)
in CH2Cl2 to give tosylate 32 in a yield of 69.4%. The FABMS
spectrum of 32 showed an peak [M + H]+ at m/z 583. Com-
pound 32 was reacted with sodium iodide to give crude com-
pound 33 (with a molecular ion peak [M]+ at m/z 538). Com-
pound 33 was reacted, without further purification, with
KSCN in DMF at 70 °C to give 26-thiocyanate 34 (38.1%
yield from 32) which showed a molecular ion peak [M]+ at
m/z 469 in the FABMS spectrum. Similarly, 33 was reacted
with KSeCN to give 26-selonocyanate 35 (24% yield from
32). The FABMS spectrum of 35 showed a molecular ion
peak [M]+ at m/z 517 (Fig. 3).
1a,3b-Diacetate 22 derived from 20 by acetylation was
reacted with N-bromosuccinimide (NBS), followed, without
further purification of the resulting mixture, by treatment with
c-collidine to give two products, (20S,22R,25R)-spirost-
1a,3b-diacetoxy-5,7-diene 24 and (20S,22R,25R)-spirost-
1a,3b-diacetoxy-4,6-diene 25 in yields of 53.0% and 4.8%,
respectively. Both products showed the same ion peak at m/z
513 [M + H]+ in the FABMS. The 13C-NMR spectrum of 24
showed four vinyl carbon signals at d 139.9, 135.2, 121.5 and
115.6 due to C-8, C-5, C-6 and C-7, respectively. Two vinyl
proton signals were observed at d 5.67 (dd, J = 6.1 and 2.7 Hz)
and 5.39 (quintet, J = 2.7 Hz) due to H-6 and H-7, respec-
1
tively, in the H-NMR spectrum of 24. Compound 25 also
showed four vinyl carbon signals at d 142.5, 131.5, 127.9 and
121.1 assignable to C-5, C-7, C-6 and C-4, respectively, in
the 13C-NMR spectrum. The 1H-NMR spectrum of 25 showed
three vinyl proton signals at d 5.98 (dd, J = 9.8 and 2.8 Hz),
5.68 (dd, J = 9.8 and 1.5 Hz) and 5.42 (broad s) due to H-6,
H-7 and H-4, respectively. Alkaline hydrolysis of 24 and 25
gave compounds 26 and 27 in yields of 92.9% and 75.8%,
respectively.
The successive irradiation and isomerization [10,19] of 5,7-
diene 26 gave the desired vitamin D derivative 12 in 20.0%
yield, which showed an ion peak at m/z 429 [M + H]+ in the
FABMS. In the 13C-NMR spectrum of 12, six vinyl carbon
signals were observed at d 147.5, 141.8, 133.6, 124.5,
117.7 and 111.8 assignable to C-10, C-8, C-5, C-6, C-7 and
1
C-19, respectively. The H-NMR spectrum of 12 exhibited
four vinyl proton signals at d 6.34 (d, J = 11.3 Hz), 6.00 (d,
J = 11.3 Hz), 5.29 (d, J = 1.8 Hz) and 4.97 (d, J = 1.8 Hz)
due to H-6, H-7, H-19a and H-19b, respectively (Table 1).
Successive bromination and dehydrobromination of diac-
etate 23 derived from 21 was performed according to the
method for the preparation of 24 to obtain (20S,22R,25R)-
spirost-1a,3a-diacetoxy-5,7-diene 28 in 25.1% yield. The
FABMS of 28 showed the same ion peak at m/z 513 [M + H]+

H.-J. Quan et al. / European Journal of Medicinal Chemistry 40 (2005) 662–673
665
Table 1
When the tosylation of 31 derived from 30 with TsCl was
performed in pyridine solution instead of CH2Cl2 solution
containing Et3N/Me3N·HCl (ca. 20:1), followed by succes-
sive reactions with NaI and KSCN, without any purification
at each step because of unisolation of the mixture at each
step, compounds 36 (m/z 469 [M + H]+, colorless foam) and
37 (m/z 429 [M]+, m.p. 201–203 °C) were obtained in 9.7%
and 12.6% yields, respectively, from 30. In the 13C-NMR
spectrum, 36 showed six vinyl carbon signals; C-22 and
C-20 on the E ring at d 151.0 and 104.6, respectively, and
C-14, C-6, C-8 and C-7 at d 144.6, 130.5, 126.2 and 124.6,
respectively. Furthermore 36 showed the carbon signal of the
SCN group at d 112.7. Compound 37 showed the carbon sig-
nal of C-22 on the spiro ring at d 109.0, and four vinyl carbon
signals at d 145.7, 130.3, 125.9 and 124.7 due to C-14, C-6,
C-8 and C-7, respectively, by HMBC. In the successive reac-
tions from 31 to 36 and 37, the 5,7-cis diene changed to the
6,8(14)-trans diene. This rearrangement may probably have
occurred during the tosylation of 31 using pyridine as cata-
lyst and solvent.
Compound 32 was treated with 3 M HCl–methanol [21]
to give compound 38 (48.9% yields), which showed an ion
peak [M + H]+ at m/z 583 in the FABMS and six vinyl car-
bon signals at 151.3, 144.6, 130.5, 126.2, 124.6 and 104.2
due to C-22, C-14, C-6, C-8, C-7 and C-20, respectively, in
the 13C-NMR spectrum. Thus, as the rearrangement from the
5,7-cis diene of 32 to the 6,8(14)-trans diene of 38 occurred
easily in 3 M HCl–methanol, it is thought that the rearrange-
ment of the cis diene of 31 in the tosylation with tosyl chlo-
ride in pyridine occurred by the action of p-toluenesulfonic
acid generated in the tosylation. Reaction of 38 with NaI,
followed by treatment with KSCN, gave compound 36 in a
yield of 75.8%.
13C-NMR spectral data of compounds provided for undergoing bioassay a
18
19
20
21
26
27
29
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
152.8 b
128.1
186.2
123.8
162.4
127.7
138.0
37.7
48.4
41.2
21.7
39.4
41.0
53.3
31.2
80.4
61.9
16.3
20.8
41.6
14.5
109.3
31.3
28.8
30.2
66.9
17.1
12
59.4
54.7
194.6
119.6
158.5
127.9
139.9
37.1
46.1
38.9
21.0
39.3
41.0
53.3
31.2
80.3
61.9
16.2
18.5
41.6
14.5
109.3
31.3
28.8
30.2
66.9
17.1
13
72.8
38.3
66.3
41.4
73.2
33.4
68.4
39.8
72.7
38.5
65.3
40.0
72.0
35.5
64.7
125.4
72.4
33.0
67.7
38.4
134.0
122.9
115.0
141.0
37.4
43.5
20.0
39.0
40.8
54.4
30.8
80.7
61.6
16.3
16.5
42.0
14.3
109.4
31.2
28.7
30.2
66.7
17.1
137.4 135.3 136.3 140.8
125.3 126.3 121.9 128.6
31.9
31.4
41.6
41.8
20.1
39.5
40.2
56.4
31.8
80.8
62.0
16.3
19.5
41.6
14.5
32.0
31.3
41.6
42.9
19.8
39.5
40.2
56.4
31.9
80.8
62.0
16.3
19.2
41.4
14.5
115.4 131.1
140.0 36.7
C-9
37.6
42.4
20.7
39.1
41.1
54.5
30.9
80.6
61.9
16.4
16.5
42.1
14.5
44.4
39.4
20.2
39.6
41.5
53.8
31.4
80.6
62.0
16.3
19.5
41.4
14.4
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
109.3 109.3 109.3 109.2
31.4
28.8
30.3
66.8
17.1
34
31.4
28.8
30.3
66.8
17.2
35
31.3
28.8
30.3
66.9
17.1
36
31.3
28.7
30.2
66.8
17.1
37
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
70.7
42.7
66.8
45.2
133.6
124.5
117.7
141.8
28.4
147.5
23.4
40.2
43.8
56.4
30.7
80.3
62.4
16.3
111.8
42.1
14.4
109.3
31.3
28.7
30.2
66.8
17.1
–
73.3
40.5
68.2
45.6
132.3
125.2
117.7
141.9
28.5
147.2
23.4
40.3
43.9
56.4
30.8
80.4
62.5
16.4
112.8
42.1
14.5
109.3
31.4
28.8
30.3
66.9
17.2
–
72.7
38.4
65.3
39.9
72.7
38.4
65.2
39.9
72.4
38.4
66.8
36.4
72.5
38.4
66.8
36.4
37.1
136.4 136.4 37.0
121.8 121.7 130.5 130.3
115.6 115.6 124.6 124.7
139.5 139.5 126.2 125.9
Irradiation of compounds 34 and 35 to obtain vitamin D
analogs having hetero atoms, such as sulfur and selenium
atoms instead of an oxygen atom, in the side ring failed
because of the formation of mixtures that could not be iso-
lated.
C-9
37.6
42.3
20.8
38.9
43.8
52.8
32.5
84.1
63.9
14.0
16.4
37.6
42.3
20.8
38.9
43.8
52.8
33.1
84.1
63.9
14.0
16.4
41.9
39.8
19.1
36.4
43.5
42.0
39.7
19.1
36.9
40.5
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
C-25
C-26
C-27
CN
144.6 145.7
3. Pharmacological results and discussion
34.5
83.8
64.0
22.2
12.3
33.0
79.6
61.3
24.1
12.3
The cytotoxic activities of synthetic vitamin D derivatives
(12 and 13), spirostanol derivatives (18–21, 26, 27, 29 and
37) and furostanol derivatives (34–36) obtained in this study
were compared with that of 1 using human colorectal (HCT
116) and human hepatoma (Hep G2) cancer cell lines. It is
known that while the former cell lines scarcely express MDR
1 (P-glycoprotein; P-gp) [22], the latter cell lines overex-
press it [23]. P-gp acts as an efflux pump to remove several
antitumor agents, Ca2+ antagonists, cyclosporine, digoxin and
other compounds from cells [24]. The cytotoxic activities of
the compounds were tested by MTT assay [25], and the IC50
values were calculated based on the percentage inhibition of
cell growth and are listed in Table 2.
104.5 104.5 104.6 43.1
11.5 11.5 11.8 14.2
150.9 150.9 151.0 109.0
33.1
23.0
33.2
41.2
18.4
33.1
23.1
33.6
37.6
19.2
32.5
23.0
33.2
41.2
18.3
31.4
28.7
30.3
67.0
17.1
–
112.8 102.0 112.7
a Spectra were obtained in CDCl3.
b Chemical shifts were in ppm from internal (CH3)4Si. Signal assignments
were based on DEPT, 1H–1H, 1H–13C COSY and 1H–13C-long-range COSY
spectral data.
Table 2 shows that in the cases of both HCT 116 and Hep
G2 cells, the IC50 values of compounds 12, 13, 18–21, 26,

666
H.-J. Quan et al. / European Journal of Medicinal Chemistry 40 (2005) 662–673
Fig. 3. Structures of compounds 30–39.
27, 29 and 34–36 were lower than those of diosgenin 1
(92.3 18.2 on HCT 116 and 21.0 7.5 µM on Hep G2),
indicating that the cytotoxic effects of the synthetic com-
pounds increased compared with 1.
differences in structures and substituents might influence the
appearance of a potent inhibitory effect on the growth of HCT
116 cells. The trans diene compound 36 having a SCN group
at the 26 position had the most potent cytotoxic effect of all
synthetic compounds on HCT 116 cells.
In the case of HCT 116, the furostanol derivatives 34 and
36 more marked effects (IC50 values: 4.9 0.3 for 34 and
1.3 0.2 µM for 36) than vitamin D derivatives 12 and 13
(IC50 values: 22.4 5.1 and 16.9 0.3 µM, respectively) and
spirostanol derivatives 18–21, 26, 27, 29 and 37 (IC50 values:
9.5 2.6–54.0 3.6 µM). Furostanol derivatives 34 and 35
had a 5,7-cis diene group and were substituted with a SCN
group and a SeCN group, respectively, at the 26 position.
Furostanol derivative 36 had a 6,8(14)-trans diene group and
was substituted with a SCN group at the 26 position. These
In the case of Hep G2 cells, no structural relationship such
as that shown in the case of HCT 116 cells was observed. In
this case, not only furostanol derivative 36 but also spirostanol
derivative 29, which had a 5,7-cis-diene group, showed simi-
lar potent cytotoxic activities (2.8 0.4 and 2.4 0.8 µM,
respectively).
Interestingly, the cytotoxic activities of all compounds syn-
thesized in this study on Hep G2 cells were more potent than
those on HCT 116 cells (Table 2) except for 34–36, although
34 and 36 showed potent activities on HCT 116 and Hep
G2 cells. Especially spirostanol derivative 29 and furostanol
derivative 36 had marked cytotoxic activities (2.4 0.8 and
2.8 0.4 µM, respectively) among the tested compounds even
on Hep G2 cells which overexpress P-gp. Furthermore,
spirostanol derivatives 20, 21, 26 and 27 and furostanol deriva-
tive 34 were very cytotoxic (5.2 1.4–7.7 1.5 µM) to Hep
G2 cells, although these derivatives were less effective than
29 and 36. These results indicate that spirostanol derivatives
20, 21, 26 and 27 and furostanol derivatives 34 and 36 as well
as 29 may be less sensitive substrates for the P-gp transport
of Hep G2 cells as far as cytotoxic activity is concerned.
Table 2
Cytotoxic effects of 1, 12, 13, 18–21, 26, 27, 29 and 34–37 on HCT 116 and
Hep G2 cells
IC50 (µM)a
Compound
1 [S]b
HCT116
Hep G2
92.3 18.2
22.4 5.1
16.9 0.3
16.0 0.4
12.3 0.5
10.5 0.5
15.1 0.4
10.4 0.3
54.0 3.6
9.5 2.6
21.0 7.5
16.0 2.1
16.3 2.9
10.3 1.8
10.3 2.8
5.2 1.4
5.9 0.6
6.8 0.5
7.7 1.5
2.4 0.8
6.4 1.2
12.7 2.2
2.8 0.4
21.6 4.7
12 [V]
13 [V]
18 [S]
19 [S]
20 [S]
21 [S]
26 [S]
27 [S]
29 [S]
34 [F]
35 [F]
36 [F]
37 [S]
Here, we attempted to clarify the intracellular target of the
compounds and the apoptosis-signaling pathway activated by
these compounds in Hep G2 cell lines. For this investigation,
different structural types of compounds 12, 26 and 36 from
vitamin D, spirostanol and furostanol derivatives, respec-
tively, were selected and investigated for their effects on the
expression of p53, Bax, p21 and Bcl-2 mRNAs. The results
are listed in Table 3.
4.9 0.3
10.4 1.0
1.3 0.2
22.7 0.6
a IC50 values (mean S.D.) are the concentrations at which 50% of the
cells are inhibited from growing.
b [S], [F] and [V] represent spirostanol, furostanol and vitamin D deriva-
tive, respectively.

H.-J. Quan et al. / European Journal of Medicinal Chemistry 40 (2005) 662–673
667
Table 3
The cytotoxic effect of diosgenin 1 on Hep G2 cells was
more potent than on HCT 116 cells. This is thought to be due
to the difference in physiological properties between Hep
G2 and HCT 116 cells.All synthesized vitamin D, spirostanol
and furostanol derivatives mentioned above showed higher
effects than 1 on both HCT 116 and Hep G2 cells. In particu-
lar, the furostanol derivatives 34 and 36 had markedly cyto-
toxic effects on HCT 116 cells, and spirostanol derivative 29
and furostanol derivative 36 had markedly potent cytotoxic
effects on Hep G2 cells. Compound 36, which had a furost-
1a,3b-dihydroxy-26-thiocyano-6,8(14),20(22)-triene struc-
ture exhibited marked effects on both HCT 116 and Hep
G2 cells. However, spirostanol 37, which had the same struc-
ture of the A to D ring as 36 was less effective than 36. In the
case of the furostanol derivatives 34–36, 6,8(14)-trans-diene
36 having a SCN group at the 26 position exhibited a greater
cytotoxic effect than the 5,7-cis-dienes 34 and 35 having a
SCN and SeCN group, respectively, at the same position, on
both HCT 116 and Hep G2 cells. Thus, the presence of the
6,8(14)-trans-diene group and a SCN group at the 26 posi-
tion in the furostanol structure may be important for produc-
ing a greater cytotoxic effect on both types of cells. Com-
pound 36 had a markedly potent cytotoxic effect on not only
HCT 116 cells weakly expressing P-pg but also P-gp overex-
pressing Hep G2 cells, which suggests that 36 is not influ-
enced by efflux systems, such as P-gp in Hep G2 cells. This
finding will be followed up by further investigations of more
potent cytotoxic compounds that are not subject to the efflux
systems of cancer cells.
Effect of derivatives 12, 26 and 36 on apoptosis-related mRNA expression
signals in Hep G2 cells
mRNA
p53
12
26
36
1.7 0.5
2.5 0.3
0.9 0.1
0.7 0.3
2.2 0.1
2.1 0.4
0.8 0.1
0.6 0.3
1.1 0.2
2.4 0.3
0.9 0.2
1.0 0.1
Bax
p21
Bcl-2
Values showed the ratios for the expression levels of these apoptosis-related
mRNA signals with drug treatment/the expression levels of control (DMSO
only). n = 3.
Table 3 shows that 12 and 26 produced significant increases
in the expression levels of apoptosis-related signals such as
p53 mRNA (1.7-fold for 12 and 2.2-fold for 26) and Bax
mRNA (2.5-fold for 12 and 2.1-fold for 26), while the expres-
sion levels of p21 mRNA (0.9-fold for 12 and 0.8-fold for
26) and Bcl-2 mRNA (0.7-fold for 12 and 0.6-fold for 26)
were unchanged, indicating that 12- and 26-induced apopto-
sis in Hep G2 cells was mediated via the activation of both
p53 and Bax. In contrast, furostanol type derivative 36 over-
expressed only Bax mRNA (2.4-fold) but not the other three
mRNAs (0.9-fold–1.1-fold).
4. Conclusions
Vitamin D type derivatives having the spiro ring and vari-
ous spirostanol and furostanol derivatives were synthesized
from 1. Successive oxidation and epoxidation of 1 gave an
important intermediate 19 which was converted to diols 20
and 21 by Birch reduction. Diacetate 22 derived from 20 was
reacted with NBS to give 1a,3b-diacetoxy-5,7-cis-diene
derivative 24 and 1a,3b-diacetoxy-4,6-trans-diene deriva-
tive 25 in 50.3% and 4.8% yields, respectively. The former
diene was deacetylated to give 26 which was successively
irradiated to obtain the desired vitamin D derivative 12 in
20.0% yield. Similarly 1a,3a-diacetoxy-5,7-cis-diene deriva-
tive 28 was obtained from diacetate 23 derived from 21. Com-
pound 28 was deacetylated to give diol 29 which was irradi-
ated to obtain vitamin D derivative 13 in 20.0% yield.
Next the synthesis of vitamin D derivatives having het-
erospiro rings such as 40 and 41 was attempted.Acetolysis of
compound 24 gave triacetate 30 which was successively
deacetylated, tosylated with Et3N/Me3N·HCl in CH2Cl2 and
iodinated to obtain iodide 33. Reaction of 33 with KSCN and
KSeCN gave furostanol derivatives 34 and 35, respectively.
Irradiation of 34 and 35 gave only a mixture that could not be
isolated, resulting in failure to obtain a vitamin D derivative
having a heterospiro ring in the side chain.
The apoptosis-signaling pathway activated by the com-
pounds in Hep G2 cell lines using the expression of p53, Bax,
p21 and Bcl-2 mRNAs, was investigated. For this investiga-
tion, three different type of compounds, vitamin D type deriva-
tive 12, spirostanol 26 and furostanol derivative 36 were
selected.
Compounds 12 and 26 showed significant increases in the
levels of apoptosis-related signals of p53 and Bax mRNA
expression, while the p21 and Bcl-2 mRNA expression
remained unchanged (Table 3). This suggests that 12- and
26-induced apoptosis in Hep G2 cells is mediated via the acti-
vation of both p53 and Bax mRNAs. These results are similar
to the report by Chresta et al. [26]; the levels of the p53 and
Bax mRNAs were increased after etoposide-treatment of the
testicular tumor line but there was no expression of the sup-
pressor of apoptosis Bcl-2 mRNA. In contrast, furostanol type
derivative 36 overexpressed only Bax mRNA but not the other
three mRNAs. Bax mRNA overexpression enhances cyto-
chrome c release from mitochondria and induces apoptosis
through a p53-independent pathway [27,28]. Takahashi et al.
[29] reported that sufficient levels of Bax mRNA may bypass
the need for upstream molecules such as p53 mRNA in the
process of chemotherapy-induced apoptosis. That report and
our own results suggest that 36 is capable of mediating cell
death in a p53-independent fashion. Perego et al. [30] reported
that mutations of the p53 gene play a role in the development
of cisplatin resistance in cancer cells. This implies that 36
The cytotoxic activities of the synthetic vitamin D deriva-
tives (12, 13), spirostanol derivatives (18–21, 26, 27, 29 and
37) and furostanol derivatives (34–36) against cancerous HCT
116 and Hep G2 cells were evaluated (Table 2). It is known
that while HCT 116 cell lines scarcely express the MDR 1
(P-glycoprotein; P-gp) [22], Hep G2 cell lines overexpress it
[23].

668
H.-J. Quan et al. / European Journal of Medicinal Chemistry 40 (2005) 662–673
may be a useful chemotherapeutical agent for the treatment
of p53-deficient and/or p53 mutant hepatoma.
ride (NH4Cl) (24 g) was added during 4 h. The cooling bath
was removed and the mixture was stirred for 2 h at room
temperature to turn white and pasty. After ammonia was
removed by a stream of argon, ethanol was slowly dropped
into the flask to decompose excessive sodium. The mixture
was poured into 5% hydrochloric acid (200 ml), and extracted
with CH2Cl2 (100 ml × 3). The organic extracts were washed
with brine (200 ml), dried over Na2SO4 and filtered. The fil-
trate was evaporated to give a residue which was applied to a
silica gel column (a gradient of 0–50% acetone in toluene) to
give 20 (1.79 g, 44.1%) as white needles, m.p. 242–244 °C
(after recrystallization from ethanol–water) (a white solid:
m.p. > 230 °C [18]) and 21 (240 mg, 5.9%) as white needles,
m.p. 245–248 °C (after recrystallization from ethanol–
5. Experimental
5.1. General procedures
Diosgenin, used as a starting material, was purchased from
SIGMA Chemical Co. Ltd., USA. Commercial p-toluene-
sulfonyl chloride (TsCl) was recrystallized from chloroform/
petroleum ether (1:5). Commercial Me3N·HCl was dried for
several minute prior to use. The other chemicals and solvents
were of reagent grade and obtained from commercial suppli-
ers. Melting points (m.p.) were determined using a
Yanagimoto micromelting point apparatus and were uncor-
rected. Kieselgel 60 F254 (E. Merck) was used for the thin-
layer chromatography (TLC). Spots were detected by spray-
ing plates with 1:9 Ce(SO4)2–10% H2SO4 reagent, followed
by heating the plate at 250 °C for 3 min. Preparative TLC
was performed on 20 × 20 cm plates coated with a 1.0 mm
layer of kieselgel 60 F254 (E. Merck). Column chromatogra-
phy was carried out using a Kieselgel 60 column (70–
230 mesh, E. Merck), and the eluates were monitored by TLC.
An SSC-6300 HPLC instrument (Senshu Scientific Co. Ltd.)
was employed for analytical HPLC (column, DOCOSIL, 10
× 250 mm; flow rate, 1.0 ml/min; column temperature, 40
°C, and DOCOSIL-B, 10 × 250 mm; flow rate, 1.0 ml/min;
column temperature, 40 °C), attached to an SSC autoinjector
6310 and an SSC fraction collector 6320 for preparative
HPLC. 1H-NMR and 13C-NMR at 500 and 125 MHz, respec-
tively, as well as 1H–1H and 1H–13C COSY, DEPT, HMBC
and NOE spectra were obtained with a JEOL JNM-A500 FT-
NMR spectrometer. Tetramethylsilane was used as an inter-
nal standard. Chemical shifts are given in ppm. Multiplicities
1
water). FABMS of 20: m/z 431 [M + H]+; H-NMR spec-
trum was agree with that of Liu et al. [18]; 13C-NMR spectral
data are listed in Table 1. HRMS (FAB) m/z calcd for
C27H43O4 [M + H]+, 431.3161, found: 431.3153. FABMS of
1
21: m/z 431 [M + H]+; H-NMR (CDCl3) (only assignable
signals are listed) d 5.58 (1H, d, J = 5.2 Hz, H-6), 4.42 (1H,
dd, J = 14.6, 7.3 Hz, H-16), 4.14 (1H, broad s, H-3b), 3.75
(1H, broad s, H-1b), 3.47 (1H, ddd, J = 11.0, 6.4, 1.8 Hz,
H-26a), 3.37 (1H, t, J = 11.0 Hz, H-26b), 0.99 (3H, s,
19-CH3), 0.97 (3H, d, J = 7.0 Hz, 21-CH3), 0.79 (3H, s,
18-CH3), 0.79 (3H, d, J = 6.1 Hz, 27-CH3); 13C-NMR spec-
tral are listed in Table 1. HRMS (FAB): m/z calcd for
C27H43O4 [M + H]+, 431.3161, found: 431.3151.
5.1.1.2. (20S,22R,25R)-Spirost-1␣,3b-diacetoxy-5-ene
(22). The general procedure for acetylation with acetic anhy-
dride and pyridine was employed for 20 (4.91 g, 11.4 mmol)
to afford 22 (5.46 g, 95.4%) as white needles, m.p. 171–173
°C (after recrystallization from ethanol–water). FABMS: m/z
515 [M + H]+. 1H-NMR (CDCl3) (only assignable signals are
listed) d 5.53 (1H, d, J = 5.5 Hz, H-6), 5.05 (1H, broad s,
H-1b), 4.91 (1H, m, H-3a), 4.41 (1H, dd, J = 15.0, 7.3 Hz,
H-16), 3.47 (1H, ddd, J = 11.0, 4.3, 1.8 Hz, H-26a), 3.37 (1H,
t, J = 11.0 Hz, H-26b), 2.05 (3H, s, COCH3), 2.02 (3H, s,
COCH3), 1.10 (3H, s, 19-CH3), 0.96 (3H, d, J = 6.7 Hz,
21-CH3), 0.79 (3H, d, J = 6.4 Hz, 27-CH3), 0.78 (3H, s,
18-CH3); 13C-NMR (CDCl3) d 170.3 (COCH3), 170.3
(COCH3), 136.1 (C-5), 124.9 (C-6), 109.3 (C-22), 80.7
(C-16), 74.5 (C-1), 69.3 (C-3), 66.8 (C-26), 62.1 (C-17), 56.4
(C-14), 42.1 (C-9), 41.6 (C-20), 40.5 (C-10), 40.3 (C-13),
39.5 (C-12), 37.3 (C-2), 31.9 (C-7), 31.8 (C-4), 31.8 (C-15),
31.4 (C-23), 31.3 (C-8), 30.3 (C-25), 28.8 (C-24), 21.3
(COCH3), 21.1 (COCH3), 20.1 (C-11), 19.4 (C-19), 17.1
(C-27), 16.3 (C-18), 14.5 (C-21). HRMS (FAB) m/z calcd for
C31H47O6 [M + H]+, 515.3373, found: 515.3360.
1
of the H-NMR signals are indicated as s (singlet), d (dou-
blet), dd (doublet of doublets), ddd (doublet of doublet of
doublets), t (triplet), quin (quintet) and m (multiplet). Fast-
atom-bombardment mass spectra (FABMS) and high resolu-
tion mass spectra (HRMS) were recorded on a JEOL JMS-DX
300 mass spectrometer.
5.1.1. Chemistry
5.1.1.1. (20S,22R,25R)-Spirost-1␣,3b-dihydroxy-5-ene (20)
and (20S,22R,25R)-spirost-1␣,3␣-dihydroxy-5-ene (21). A
three-necked flask connected with a dropping funnel, a sealed
mechanical stirrer and an inlet tube connected to an ammo-
nia source was fitted in a cooling bath with liquid nitrogen in
ethanol.After swepting with a stream of argon gas for 10 min,
ammonia (160 ml) was trapped in the flask, then sodium
pieces (10 g) was added under stirring. After further stirring
for 20 min, the epoxide 19 (4.0 g, 9.4 mmol) in THF (90 ml)
was added dropwise during 40 min. The cooling bath was
removed and the mixture was stirred for 1 h. The flask was
dipped into the cooling bath, and anhydrous ammonium chlo-
5.1.1.3. (20S,22R,25R)-Spirost-1␣,3␣-diacetoxy-5-ene
(23). The general procedure for acetylation with acetic anhy-
dride and pyridine was employed for 21 (1.60 g, 3.72 mmol)
to give 23 (1.50 g, 78.4%) as white needles, m.p. 191–193 °C
(after recrystallization from acetone–water). FABMS: m/z 515
1
[M + H]+; H-NMR (CDCl3) (only assignable signals are

H.-J. Quan et al. / European Journal of Medicinal Chemistry 40 (2005) 662–673
669
listed) d 5.47 (1H, d, J = 5.5 Hz, H-6), 5.00 (1H, broad s,
H-3b), 4.89 (1H, broad s, H-1b), 4.42 (1H, dd, J = 15.0,
7.6 Hz, H-16), 3.47 (1H, ddd, J = 11.0, 4.6, 2.1 Hz, H-26a),
3.37 (1H, t, J = 11.0 Hz, H-26b), 2.03 (3H, s, COCH3), 2.01
(3H, s, COCH3), 1.08 (3H, s, 19-CH3), 0.96 (3H, d, J = 7.0 Hz,
21-CH3), 0.79 (3H, d, J = 7.9 Hz, 27-CH3), 0.78 (3H, s,
18-CH3); 13C-NMR(CDCl3) d 170.4 (COCH3), 170.4
(COCH3), 134.5 (C-5), 124.5 (C-6), 109.2 (C-22), 80.7
(C-16), 73.1 (C-1), 68.6 (C-3), 66.8 (C-26), 62.0 (C-17), 56.4
(C-14), 41.8 (C-9), 41.5 (C-20), 40.6 (C-10), 40.2 (C-13),
39.5 (C-12), 35.4 (C-4), 31.8 (C-7), 31.8 (C-15), 31.3 (C-23),
31.2 (C-8), 30.2 (C-25), 29.1 (C-2), 28.7 (C-24), 21.4
(COCH3), 21.2 (COCH3), 19.8 (C-11), 19.3 (C-19), 17.1
(C-27), 16.2 (C-18), 14.5 (C-21). HRMS (FAB): m/z calcd
for C31H47O6 [M + H]+, 515.3373, found: 515.3363.
3.37 (1H, t, J = 11.0 Hz, H-26b), 2.06 (3H, s, COCH3), 2.03
(3H, s, COCH3), 1.09 (3H, s, 19-CH3), 0.96 (3H, d, J = 7.0 Hz,
21-CH3), 0.84 (3H, s, 18-CH3), 0.79 (3H, d, J = 6.1 Hz,
27-CH3); 13C-NMR(CDCl3) d 170.7 (COCH3), 170.6
(COCH3), 142.5 (C-5), 131.5 (C-7), 127.9 (C-6), 121.1 (C-4),
109.2 (C-22), 80.5 (C-16), 73.9 (C-1), 68.0 (C-3), 66.8 (C-26),
62.0 (C-17), 53.8 (C-14), 44.5 (C-9), 41.5 (C-13), 41.4 (C-20),
39.6 (C-12), 38.2 (C-10), 36.7 (C-8), 31.3 (C-15), 31.3 (C-23),
30.2 (C-25), 28.7 (C-24), 28.4 (C-2), 21.2 (COCH3), 21.0
(COCH3), 20.1 (C-11), 19.0 (C-19), 17.1 (C-27), 16.3 (C-18),
14.4 (C-21). HRMS (FAB): m/z calcd for C31H45O6 [M + H]+,
513.3216, found: 513.3215.
5.1.1.5. (20S,22R,25R)-Spirost-1␣,3b-dihydroxy-5,7-diene
(26). A solution of compound 24 (270 mg, 0.53 mmol) in
MeOH (8 ml) and benzene (9 ml) was combined with metha-
nolic 2 M KOH (10 ml). The mixture was stirred at 65 °C for
1 h, and then poured into ice-cold water (100 ml) and extracted
with CH2Cl2 (70 ml × 3). The organic extracts were washed
with brine (150 ml), dried and evaporated to give a residue
which was recrystallized from ethanol–water to afford 26
(210 mg, 92.9%) as white needles (m.p. 233–234 °C, recrys-
5.1.1.4. (20S,22R,25R)-Spirost-1␣,3b-diacetoxy-5,7-diene
(24) and (20S,22R,25R)-spirost-1␣,3b-diacetoxy-4,6-diene
(25). A mixture of diacetate 22 (1.10 g, 2.14 mmol) and
N-bromosuccinimide (NBS) (495 mg, 2.78 mmol) in anhy-
drous n-hexane (50 ml) was refluxed for 2 h. After cooling to
room temperature, the reaction mixture was filtered. The fil-
trate was evaporated to give a residue which was dissolved in
xylene (50 ml). The mixture was combined with c-collidine
(8 ml) and refluxed for 2 h, and then filtered. The filtrate was
diluted with diethyl ether (Et2O) (200 ml). The mixture was
washed with 5% aqueous HCl (100 ml) and brine (100 ml),
dried over Na2SO4, and filtered. The filtrate was evaporated
to give a residue which was subjected to silica gel column
chromatography (a gradient of 0–10% ethyl acetate in
n-hexane) to afford 24 (581 mg, 53.0%) as white needles,
m.p. 195–196 °C (after recrystallization from acetone) and
25 (52 mg, 4.8%) as white needles, m.p. 152–154 °C (after
recrystallization from acetone–H2O). FABMS of 24: m/z 513
1
tallizaion from acetone). FABMS: m/z 429 [M + H]+; H-
NMR (CDCl3) (only assignable signals are listed) d 5.70 (1H,
dd, J = 5.8, 2.4 Hz, H-6), 5.37 (1H, quin, J = 2.4 Hz, H-7),
4.50 (1H, dd, J = 14.5, 7.6 Hz, H-16), 4.05 (1H, m, H-3a),
3.75 (1H, broad s, H-1b), 3.48 (1H, ddd, J = 11.0, 4.3, 2.1 Hz,
H-26a), 3.39 (1H, t, J = 11.0 Hz, H-26b), 2.73 (1H, m, H-9),
0.99 (3H, d, J = 6.7 Hz, 21-CH3), 0.95 (3H, s, 19-CH3), 0.80
(3H, d, J = 6.4 Hz, 27-CH3), 0.74 (3H, s, 18-CH3); 13C-
NMR spectral data are listed in Table 1. HRMS (FAB): m/z
calcd for C27H41O4 [M + H]+, 429.3005, found: 429.2977.
5.1.1.6. (20S,22R,25R)-Spirost-1␣,3b-dihydroxy-4,6-diene
(27). The same reaction of 4,6-dien diacetate 25 (64 mg,
0.12 mmol) as described for 26 gave 27 (42 mg, 75.8%) as
white needles (m.p. 208–210 °C, recrystallization from
1
[M + H]+; H-NMR (CDCl3) (only assignable signals are
listed) d 5.67 (1H, dd, J = 6.1, 2.7 Hz, H-6), 5.39 (1H, quin,
J = 2.7 Hz, H-7), 4.99 (1H, broad s, H-1b), 4.99 (1H, m,
H-3a), 4.51 (1H, dd, J = 14.3, 7.6 Hz, H-16), 3.48 (1H, ddd,
J = 11.0, 4.3, 1.8 Hz, H-26a), 3.38 (1H, t, J = 11.0 Hz, H-26b),
2.43 (1H, m, H-9), 2.09 (3H, s, COCH3), 2.04 (3H, s,
COCH3), 1.03 (3H, s, 19-CH3), 0.98 (3H, d, J = 6.7 Hz,
21-CH3), 0.80 (3H, d, J = 6.4 Hz, 27-CH3), 0.72 (3H, s,
18-CH3); 13C-NMR(CDCl3) d 170.4 (COCH3), 170.3
(COCH3), 139.9 (C-8), 135.2 (C-5), 121.5 (C-6), 115.6 (C-7),
109.3 (C-22), 80.5 (C-16), 74.2 (C-1), 68.4 (C-3), 66.9 (C-26),
61.9 (C-17), 54.4 (C-14), 42.1 (C-20), 41.1 (C-10), 40.9
(C-13), 38.9 (C-12), 37.7 (C-9), 35.8 (C-2), 32.0 (C-4), 31.4
(C-23), 30.9 (C-15), 30.3 (C-25), 28.8 (C-24), 21.3 (COCH3),
21.2 (COCH3), 20.1 (C-11), 17.1 (C-27), 16.4 (C-18), 16.2
(C-19), 14.5 (C-21); HRMS (FAB) m/z calcd for C31H45O6
[M + H]+, 513.3216, found: 513.3205. FABMS of 25: m/z
513 [M + H]+; 1H-NMR (CDCl3) (only assignable signals are
listed) d 5.98 (1H, dd, J = 9.8, 2.8 Hz, H-6), 5.68 (1H, dd,
J = 9.8, 1.5 Hz, H-7), 5.43 (1H, broad s, H-3a), 5.42 (1H,
broad s, H-4), 5.03 (1H, broad s, H-1b), 4.43 (1H, dd, J = 14.7,
7.6 Hz, H-16), 3.47 (1H, ddd, J = 11.0, 4.0, 1.5 Hz, H-26a),
1
acetone). FABMS: m/z 429 [M + H]+; H-NMR (CDCl3)
(only assignable signals are listed) d 5.97 (1H, dd, J = 9.8,
2.4 Hz, H-6), 5.64 (1H, d, J = 9.8 Hz, H-7), 5.48 (1H, broad
s, H-4), 4.52 (1H, m, H-3a), 4.43 (1H, dd, J = 15.0, 7.6 Hz,
H-16), 3.89 (1H, broad s, H-1b), 3.47 (1H, ddd, J = 11.0, 4.3,
1.5 Hz, H-26a), 3.37 (1H, t, J = 11.0 Hz, H-26b), 1.02 (3H, s,
19-CH3), 0.97 (3H, d, J = 7.0 Hz, 21-CH3), 0.85 (3H, s,
18-CH3), 0.79 (3H, d, J = 6.4 Hz, 27-CH3); 13C-NMR spec-
tral date are listed in Table 1. HRMS (FAB): m/z calcd for
C27H41O4 [M + H]+, 429.3005, found: 429.2998.
5.1.1.7. (20S,22R,25R)-Spirost-1␣,3␣-diacetoxy-5,7-diene
(28). The same reaction and procedure for 23 (100 mg,
0.194 mmol) as described for 24 gave 28 (21 mg, 25.1%) as a
white needles, m.p. 152–155 °C (after recrystallization from
acetone–H2O). FABMS: m/z 513 [M + H]+; 1H-NMR
(CDCl3) (only assignable signals are listed) d 5.68 (1H, dd,
J = 5.5, 2.4 Hz, H-6), 5.43 (1H, quin, J = 2.4 Hz, H-7), 5.08
(1H, broad s, H-3b), 4.89 (1H, m, H-1b), 4.51 (1H, dd,

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H.-J. Quan et al. / European Journal of Medicinal Chemistry 40 (2005) 662–673
J = 14.3, 7.6 Hz, H-16), 3.49 (1H, ddd, J = 11.0, 4.6, 1.8 Hz,
H-26a), 3.39 (1H, t, J = 11.0 Hz, H-26b), 2.57 (1H, m, H-9),
2.07 (3H, s, COCH3), 2.02 (3H, s, COCH3), 1.02 (3H, s,
19-CH3), 0.99 (3H, d, J = 6.7 Hz, 21-CH3), 0.80 (3H, d,
J = 6.4 Hz, 27-CH3), 0.72 (3H, s, 18-CH3); 13C-NMR(CDCl3)
d 170.5 (COCH3), 170.4 (COCH3), 139.9 (C-8), 134.2 (C-5),
121.2 (C-6), 115.8 (C-7), 109.3 (C-22), 80.5 (C-16), 72.5
(C-1), 67.7 (C-3), 66.8 (C-26), 61.9 (C-17), 54.5 (C-14), 42.0
(C-20), 41.0 (C-10), 40.9 (C-13), 38.8 (C-12), 37.7 (C-9),
34.1 (C-4), 31.3 (C-23), 30.8 (C-15), 30.2 (C-25), 30.0 (C-2),
28.8 (C-24), 21.4 (COCH3), 21.3 (COCH3), 20.0 (C-11), 17.1
(C-27), 16.4 (C-19), 16.3 (C-18), 14.5 (C-21). HRMS (FAB):
m/z calcd for C31H45O6 [M + H]+, 513.3216, found: 513.3199.
3.38 (1H, t, J = 11.0 Hz, H-26b), 0.98 (3H, d, J = 7.0 Hz,
21-CH3), 0.79 (3H, d, J = 6.4 Hz, 27-CH3), 0.66 (3H, s,
18-CH3); 13C-NMR spectral data are listed in Table 1. HRMS
(FAB): m/z calcd for C27H41O4 [M + H]+, 429.3005, found:
429.2987.
5.1.1.11. (25R)-Furost-1␣,3b,26-triacetoxy-5,7,20(22)-
triene (30). A solution of 24 (927 mg, 1.81 mmol) in acetic
anhydride (30 ml) was heated in a bomb tube at 180 °C for
8 h. The reaction mixture was coevaporated with toluene to
afford a residue which was subjected to column chromatog-
raphy (a gradient of 0–10% ethyl acetate in n-hexane) to give
compound 30 (785 mg, 78.3%) as a colorless foam. FABMS:
1
m/z 555 [M + H]+; H-NMR (CDCl3) (only assignable sig-
5.1.1.8. (20S,22R,25R)-Spirost-1␣,3␣-dihydroxy-5,7-diene
(29). The reaction of 28 (210 mg, 0.41 mmol) as described
for 26 gave 29 (142 mg, 80.9%) as white needles, m.p. 212–
214 °C (recrystallization from acetone). FABMS: m/z 429
nals are listed) d 5.68 (1H, dd, J = 5.5, 2.1 Hz, H-6), 5.43
(1H, quin, J = 2.1 Hz, H-7), 5.00 (1H, brs, H-1b), 5.00 (1H,
m, H-3a), 4.83 (1H, ddd, J = 10.1, 7.9, 5.8 Hz, H-16), 3.94
(1H, dd, J = 10.7, 5.8 Hz, H-26a), 3.88 (1H, dd, J = 10.7,
6.4 Hz, H-26b), 2.61 (1H, d, J = 10.1 Hz, H-17), 2.44 (1H,
m, H-9), 2.09 (3H, COCH3), 2.05 (3H, COCH3), 2.04 (3H,
COCH3), 1.59 (3H, s, 21-CH3), 1.03 (3H, s, 19-CH3), 0.94
(3H, d, J = 6.7 Hz, 27-CH3), 0.63 (3H, s, 18-CH3); 13C-
NMR(CDCl3) d 171.3 (COCH3), 170.4 (COCH3), 170.3
(COCH3), 151.8 (C-22), 139.5 (C-8), 135.4 (C-5), 121.5
(C-6), 115.9 (C-7), 103.9 (C-20), 84.0 (C-16), 74.3 (C-1),
69.2 (C-26), 68.4 (C-3), 64.0 (C-17), 52.8 (C-14), 43.7 (C-13),
41.1 (C-10), 38.8 (C-12), 37.8 (C-9), 35.8 (C-2), 33.2 (C-23),
32.1 (C-25), 32.0 (C-4), 30.8 (C-15), 23.2 (C-24), 21.3
(COCH3), 21.2 (COCH3), 21.0 (COCH3), 20.3 (C-11), 16.7
(C-27), 16.2 (C-19), 14.0 (C-18), 11.5 (C-21). HRMS (FAB)
m/z calcd for C33H47O7 [M + H]+: 555.3322, found: 555.3307.
1
[M + H]+; H-NMR (CDCl3) (only assignable signals are
listed) d 5.71 (1H, dd, J = 5.5, 2.4 Hz, H-6), 5.37 (1H, quin,
J = 2.4 Hz, H-7), 4.51 (1H, dd, J = 14.6, 7.3 Hz, H-16), 4.23
(1H, broad s, H-3b), 3.63 (1H, broad s, H-1b), 3.49 (1H, ddd,
J = 11.0, 4.6, 1.8 Hz, H-26a), 3.40 (1H, t, J = 11.0 Hz, H-26b),
3.06 (1H, m, H-9), 0.98 (3H, d, J = 6.7 Hz, 21-CH3), 0.92
(3H, s, 19-CH3), 0.80 (3H, d, J = 7.1 Hz, 27-CH3), 0.72 (3H,
s, 18-CH3); 13C-NMR spectral data are listed in Table 1.
HRMS (FAB): m/z calcd for C27H41O4 [M + H]+, 429.3005,
found: 429.2997.
5.1.1.9. (20S,22R,25R)-9,10-Secospirosta-5,7,10(19)-triene-
1␣,3b-diol (12). Irradiation of 26 (120 mg, 0.28 mmol) in
Et2O (600 ml) with a 200 W high pressure mercury lamp
(UV-LT10) (Ishii Sho-ten Co., Ltd., Tokyo, Japan) through a
Pyrex filter (No. 7740) was performed according to the
method reported by Morisaki et al. [10] to give 12 (24 mg,
20.0%) as a colorless foam. FABMS: m/z 429 [M + H]+;
1H-NMR (CDCl3) (only assignable signals are listed) d 6.34
(1H, d, J = 11.3 Hz, H-6), 6.00 (1H, d, J = 11.3 Hz, H-7),
5.29 (1H, d, J = 1.8 Hz, H-19a), 4.97 (1H, d, J = 1.8 Hz,
H-19b), 4.48 (1H, dd, J = 15.0, 7.3 Hz, H-16), 4.42 (1H, dd,
J = 7.3, 4.3 Hz, H-1b), 4.23 (1H, m, H-3a), 3.48 (1H, ddd,
J = 11.0, 4.3, 2.1 Hz, H-26a), 3.38 (1H, t, J = 11.0 Hz, H-26b),
0.98 (3H, d, J = 7.0 Hz, 21-CH3), 0.79 (3H, d, J = 6.4 Hz,
27-CH3), 0.67 (3H, s, 18-CH3); 13C-NMR spectral data are
listed in Table 1. HRMS (FAB): m/z calcd for C27H41O4
[M + H]+, 429.3005, found: 429.2990.
5.1.1.12. (25R)-Furost-1␣,3b-dihydroxy-26-p-toluenesulfo-
nyloxy-5,7,20(22)-triene (32). A solution of triene acetate 30
(280 mg, 0.51 mmol) in methanolic 2 M KOH solution (8 ml)
was stirred at room temperature for 1 h. Then the reaction
mixture was poured into 5% NaHCO3 aqueous solution
(80 ml) and extracted with CH2Cl2 (50 ml × 3). The extracts
were washed with brine (150 ml), dried over Na2SO4 and
filtered. The filtrate was evaporated to give a crude triol 31
(213 mg). FABMS: m/z 429 [M + H]+; HRMS (FAB) m/z
calcd for C27H41O4 [M + H]+, 429.3005, found: 429.2993. A
solution of 31 (213 mg) in anhydride CH2Cl2 (4.6 ml) was
added to a stirred solution of Et3N (0.14 ml, 1.02 mmol),
Me3N.HCl (5 mg, 0.05 mmol) and p-toluenesulfonylchloride
(TsCl) (116 mg, 0.61 mmol) in anhydrous CH2Cl2 (1 ml) at
0 °C, and the mixture was stirred for 10 min. Ice-cold water
(80 ml) was added to the mixture, followed by extraction with
CH2Cl2 (50 ml × 3). The organic extracts were washed suc-
cessively with 5% HCl (150 ml), 5% NaHCO3 (100 ml) and
brine (200 ml), dried over Na2SO4 and filtered. The filtrate
was evaporated to give a residue which was purified by silica
gel chromatography (a gradient of 0–25% acetone in tolu-
ene) to obtain the tosylate 32 (204 mg, 69.4%) as a yellowish
foam. FABMS: m/z 583 [M + H]+; 1H-NMR (CDCl3) (only
assignable signals are listed) d 7.78 (2H, d, J = 7.9 Hz, C6H4),
5.1.1.10. (20S,22R,25R)-9,10-Secospirosta-5,7,10(19)-triene-
1␣,3␣-diol (13). The same reaction of 29 (50 mg, 0.12 mmol)
as described for 12 gave 13 (10 mg, 20.0%) as a colorless
foam. FABMS: m/z 429 [M + H]+; 1H-NMR (CDCl3) (only
assignable signals are listed) d 6.41 (1H, d, J = 11.3 Hz, H-6),
6.02 (1H, d, J = 11.3 Hz, H-7), 5.25 (1H, d, J = 1.8 Hz,
H-19a), 4.97 (1H, d, J = 1.8 Hz, H-19b), 4.49 (1H, dd,
J = 15.0, 7.3 Hz, H-16), 4.32 (1H, broad s, H-1b), 4.05 (1H,
broad s, H-3b), 3.48 (1H, ddd, J = 11.0, 4.3, 2.1 Hz, H-26a),

H.-J. Quan et al. / European Journal of Medicinal Chemistry 40 (2005) 662–673
671
7.34 (2H, d, J = 7.9 Hz, C6H4), 5.70 (1H, dd, J = 5.5, 1.8 Hz,
H-6), 5.40 (1H, quin, J = 1.8 Hz, H-7), 4.78 (1H, ddd,
J = 10.1, 7.6, 5.8 Hz, H-16), 4.06 (1H, m, H-3a), 3.89 (1H,
dd, J = 9.5, 5.2 Hz, H-26a), 3.80 (1H, dd, J = 9.5, 6.7 Hz,
H-26b), 3.76 (1H, broad s, H-1b), 2.75 (1H, m, H-9), 2.58
(1H, d, J = 10.1 Hz, H-17), 2.44 (3H, s, C6H4-CH3), 1.54
(3H, s, 21-CH3), 0.95 (3H, s, 19-CH3), 0.90 (3H, d, J = 6.7 Hz,
27-CH3), 0.60 (3H, s, 18-CH3); 13C-NMR(CDCl3) d 151.2
(C-22), 144.6 (a phenyl carbon), 139.5 (C-8), 136.5 (C-5),
133.0, 129.8 and 127.8 (phenyl carbons), 121.7 (C-6), 115.6
(C-7), 104.2 (C-20), 84.0 (C-16), 74.8 (C-26), 72.6 (C-1),
65.2 (C-3), 63.9 (C-17), 52.8 (C-14), 43.8 (C-13), 42.3 (C-10),
39.9 (C-4), 38.9 (C-12), 38.4 (C-2), 37.6 (C-9), 33.1 (C-23),
32.3 (C-25), 30.0 (C-15), 22.9 (C-24), 21.6 (C6H4–CH3), 20.8
(C-11), 16.4 (C-27), 16.3 (C-19), 13.9 (C-18), 11.4 (C-21).
HRMS (FAB) m/z calcd for C34H47O6S [M + H]+, 583.3093,
found: 583.3110.
5.1.1.14. (25R)-Furost-1␣,3b-dihydroxy-26-selenocyano-
5,7,20(22)-triene (35). The similar reaction of crude 33
(174 mg, 0.78 mmol) derived from 32 with KSeCN (93 mg,
0.64 mmol) described above gave compound 35 (40 mg, 24%)
1
as a colorless foam. FABMS: m/z 517 [M]+; H-NMR
(CDCl3) (only assignable signals are listed) d 5.71 (1H, dd,
J = 5.5, 2.1 Hz, H-6), 5.41 (1H, quin, J = 2.1 Hz, H-7), 4.84
(1H, ddd, J = 10.1, 7.6, 5.5 Hz, H-16), 4.05 (1H, m, H-3a),
3.76 (1H, broad s, H-1b), 3.16 (1H, dd, J = 11.9, 5.5 Hz,
H-26a), 2.96 (1H, dd, J = 11.9, 7.3 Hz, H-26b), 2.75 (1H, m,
H-9), 2.63 (1H, d, J = 10.1 Hz, H-17), 1.61 (3H, s, 21-CH3),
1.08 (3H, d, J = 6.7 Hz, 27-CH3), 0.95 (3H, s, 19-CH3), 0.64
(3H, s, 18-CH3); 13C-NMR spectral data are listed in Table 1.
HRMS (FAB) m/z calcd for C28H39O3NSe [M]+, 517.2095,
found: 517.2098.
5.1.1.15. (25R)-Furost-1␣,3b-dihydroxy-26-thiocyano-
6,8(14),20(22)-triene (36) and (20S,22R,25R)-spirost-1␣,3b-
dihydroxy-6,8(14)-diene (37). A solution of crude 31 (378 mg,
0.83 mmol) derived from 30 in pyridine (8 ml) was added
with TsCl (473 mg, 2.49 mmol) in pyridine (3 ml), then stirred
at 0 °C for 1.5 h. The mixture was poured into ice-cold water
(100 ml) and extracted with CH2Cl2 (80 ml × 3). The organic
extracts were washed with 5% HCl (100 ml × 3), 5% NaHCO3
solution (200 ml) and brine (250 ml), dried over Na2SO4 and
filtered. The filtrate was evaporated to give a residue (363 mg)
which was dissolved in 3-pentanone (12 ml) and mixed with
NaI (187 mg, 1.25 mmol). The mixture was stirred at 70 °C
for 5 h. The reaction mixture was then poured into ice-cold
water (50 ml) and extracted with CH2Cl2 (30 ml × 3). The
organic extracts were washed with 10% Na2S2O3 aqueous
solution (80 ml × 2) and brine (100 ml), and dried over anhy-
drous Na2SO4. After filtration, the filtrate was evaporated to
give a crude iodide (342 mg). A solution of the crude iodide
(342 mg, 0.64 mmol) in DMF (20 ml) was mixed with KSCN
(124 mg, 1.28 mmol), and the mixture was stirred at 70 °C
for 2 h. The reaction mixture was poured into ice-water
(50 ml), and extracted with CH2Cl2 (50 ml × 3). The organic
extracts were washed with brine (150 ml) and dried over
Na2SO4. After filtration, the filtrate was evaporated to give a
residue which was subjected to preparative HPLC (30% H2O–
acetone) to give compounds 36 (colorless foam, 38 mg, 9.7%,
from 30) and 37 (white needles, m.p. 201–203 °C, after recrys-
tallization from acetone, 45 mg, 12.6%, from 30). FABMS of
5.1.1.13. (25R)-Furost-1␣,3b-dihydroxy-26-thiocyano-
5,7,20(22)-triene (34). Sodium iodide (227 mg, 1.51 mmol)
was added to a solution of tosylate 32 (440 mg, 0.76 mmol)
in 3-pentanone (14 ml), and the mixture was stirred at 70 °C
for 7 h. The reaction mixture was poured into ice-cold water
(100 ml), and extracted with CH2Cl2 (80 ml × 3). The organic
extracts were washed with 10% Na2S2O3 aqueous solution
(100 ml × 2) and brine (200 ml), and dried over anhydrous
Na2SO4. After filtration, the filtrate was evaporated under
vacuum to give crude compound 33 (419 mg). FABMS: m/z
1
538 [M]+; H-NMR (CDCl3) (only assignable signals are
listed) d 5.73 (1H, dd, J = 5.8, 2.8 Hz, H-6), 5.42 (1H, quin,
J = 2.8 Hz, H-7), 4.84 (1H, ddd, J = 10.1, 7.9, 5.8 Hz, H-16),
4.07 (1H, m, H-3a), 3.78 (1H, broad s, H-1b), 3.25 (1H, dd,
J = 9.8, 4.3 Hz, H-26a), 3.16 (1H, dd, J = 9.8, 5.8 Hz, H-26b),
2.62 (1H, d, J = 10.1 Hz, H-17), 1.61 (3H, s, 21-CH3), 0.99
(3H, d, J = 6.4 Hz, 27-CH3), 0.97 (3H, s, 19-CH3), 0.65 (3H,
s, 18-CH3); HRMS (FAB) m/z calcd for C27H39O3I [M]+,
538.1944, found: 538.1935. To a solution of crude 33 (419 mg,
0.78 mmol) in DMF (22 ml) was added KSCN (152 mg,
1.56 mmol), and the mixture was stirred at 70 °C for 1.5 h.
The reaction mixture was poured into ice-water (100 ml), and
extracted with CH2Cl2 (80 ml × 3). The organic extracts were
washed with brine (200 ml) and dried over Na2SO4. After
filtration, the filtrate was evaporated under vacuum to give a
residue which was purified by preparative HPLC (35% H2O–
acetone) to afford compound 34 (135 mg, 38.1% from 32) as
a colorless foam. FABMS: m/z 469 [M]+; 1H-NMR (CDCl3)
(only assignable signals are listed) d 5.71 (1H, dd, J = 5.5,
2.1 Hz, H-6), 5.41 (1H, quin, J = 2.1 Hz, H-7), 4.84 (1H, ddd,
J = 10.1, 7.6, 5.8 Hz, H-16), 4.06 (1H, m, H-3a), 3.76 (1H,
broad s, H-1b), 3.03 (1H, dd, J = 12.8, 5.5 Hz, H-26a), 2.80
(1H, dd, J = 12.8, 7.6 Hz, H-26b), 2.75 (1H, m, H-9), 2.63
(1H, d, J = 10.1 Hz, H-17), 1.61 (3H, s, 21-CH3), 1.07 (3H,
d, J = 6.7 Hz, 27-CH3), 0.95 (3H, s, 19-CH3), 0.64 (3H, s,
18-CH3); 13C-NMR spectral data are listed in Table 1. HRMS
(FAB) m/z calcd for C28H39O3NS [M]+, 469.2651, found:
469.2658.
1
36: m/z 469 [M]+; H-NMR (CDCl3) (only assignable sig-
nals are listed) d 6.12 (1H, dd, J = 9.8, 3.1 Hz, H-7), 5.35
(1H, dd, J = 9.8, 1.6 Hz, H-6), 4.82 (1H, ddd, J = 10.1, 8.2,
4.6 Hz, H-16), 4.08 (1H, m, H-3a), 3.90 (1H, broad s, H-1b),
3.04 (1H, dd, J = 12.8, 5.5 Hz, H-26a), 2.81 (1H, dd, J = 12.8,
7.6 Hz, H-26b), 2.67 (1H, d, J = 14.0 Hz, H-5), 2.59 (1H, d,
J = 10.1 Hz, H-17), 1.63 (3H, s, 21-CH3), 1.08 (3H, d,
J = 6.7 Hz, 27-CH3), 0.95 (3H, s, 18-CH3), 0.67 (3H, s,
19-CH3); 13C-NMR spectral data are listed in Table 1. HRMS
(FAB) of 36 m/z calcd for C28H39O3NS [M]+, 469.2651,
found: 469.2645. FABMS of 37: m/z 429 [M + H]+; 1H-NMR
(CDCl3) (only assignable signals were listed) d 6.12 (1H, dd,

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H.-J. Quan et al. / European Journal of Medicinal Chemistry 40 (2005) 662–673
J = 9.8, 3.1 Hz, H-7), 5.33 (1H, dd, J = 9.8, 1.5 Hz, H-6),
4.38 (1H, dd, J = 15.0, 7.6 Hz, H-16), 4.08 (1H, m, H-3a),
3.89 (1H, broad s, H-1b), 3.48 (1H, ddd, J = 10.7, 4.0, 1.8 Hz,
H-26a), 3.39 (1H, t, J = 10.7 Hz, H-26b), 1.04 (3H, s,
18-CH3), 0.99 (3H, d, J = 6.4 Hz, 21-CH3), 0.80 (3H, d,
J = 6.3 Hz, 27-CH3), 0.67 (3H, s, 19-CH3); 13C-NMR spec-
tral data are listed in Table 1. HRMS (FAB) of 37 m/z calcd
for C27H41O4 [M + H]+, 429.3005, found: 429.2980.
co’s modified Eagle’s medium (DMEM), McCoy’s 5A
medium, fetal bovine serum (FBS) and penicillin–streptomy-
cin mixture (100 U/ml penicillin and 100 µg/ml streptomy-
cin) were purchased from Wako Pure Chemical Industries,
Ltd. (Osaka, Japan), Sigma (MO, USA), Biosource Interna-
tional (CA, USA) and Bio Whittaker (ND, USA), respec-
tively. The HCT 116 cells were maintained in McCoy’s 5A
medium and Hep G2 cells were cultured in DMEM. Each
medium was supplemented with 10% FBS and a penicillin–
streptomycin mixture at 37 °C in a humidified atmosphere
containing 5% CO2 in air.
5.1.1.16. (25R)-Furost-1␣,3b-dihydroxy-26-p-toluenesulfo-
nyloxy -6,8(14),20(22)-triene (38). To a solution of the 5,7-
diene tosylate 32 (45 mg, 0.08 mmol) in chloroform (2 ml)
was added 3 M HCl–methanol (8 ml) followed by stirring at
room temperature for 20 h. Then the reaction mixture was
poured into 5% NaHCO3 aqueous solution (30 ml), and
extracted with CH2Cl2 (30 ml × 3). The extracts were washed
with brine (100 ml), dried over Na2SO4 and evaporated to
give a residue which was purified by silica gel column chro-
matography (a gradient of 0–15% acetone in toluene) to afford
38 (22 mg, 48.9%) as a colorless foam. FABMS: m/z 583
5.1.2.2. Cytotoxicity test. Aliquots (200 µl) of 5 × 103 cells
per ml of HCT 116 and Hep G2 cells were seeded in 96 well
flat-bottomed plates (MicrotestTM Tissue Culture Plate,
96 Well, Flat Bottom with Low Evaporation Ltd., Falcon, NJ,
USA), and were incubated in a medium containing 10% FBS
and a penicillin–streptomycin mixture at 37 °C in a humidi-
fied atmosphere of 5% CO2 for 24 h. The test drugs were
dissolved in dimethyl sulfoxide (DMSO). The incubation
medium was replaced with each test medium giving a final
concentration of 1–500 µmol/l of test compounds and no drug
in 2 µl DMSO over 2 days.
The ability of the drug to inhibit cellular growth was deter-
mined using the MTT assay [25]. The cytotoxic activities of
the test drugs were determined as previously described [31].
Each experiment was performed in duplicate wells, and all
experiments involving control (DMSO only) and the drug
treatments were performed separately three to five times. Data
represent mean S.D. values.
1
[M + H]+; H-NMR (CDCl3) (only assignable signals are
listed) d 7.78 (2H, d, J = 7.9 Hz, C6H4), 7.34 (2H, d,
J = 7.9 Hz, C6H4), 6.12 (1H, dd, J = 9.8, 3.4 Hz, H-7), 5.33
(1H, dd, J = 9.8, 1.5 Hz, H-6), 4.75 (1H, ddd, J = 10.1, 7.6,
5.8 Hz, H-16), 4.09 (1H, m, H-3b), 3.90 (1H, dd, J = 9.5,
4.9 Hz, H-26a), 3.89 (1H, broad s, H-1a), 3.81 (1H, dd,
J = 9.5, 6.4 Hz, H-26b), 2.64 (1H, d, J = 15.0 Hz, H-5), 2.59
(1H, d, J = 10.1 Hz, H -17), 2.44 (3H, s, C6H4-CH3), 1.56
(3H, s, 21-CH3), 0.91 (3H, s, 18-CH3), 0.91 (3H, d, J = 6.7 Hz,
27-CH3), 0.67 (3H, s, 19-CH3); 13C-NMR(CDCl3) d 151.3
(C-22), 144.6 (C-14), 144.6 and 133.1 (phenyl carbons), 130.5
(C-6), 129.8 and 127.8 (phenyl carbons), 126.2 (C-8), 124.6
(C-7), 104.2 (C-20), 83.7 (C-16), 74.8 (C-26), 72.4 (C-1),
66.8 (C-3), 64.0 (C-17), 43.5 (C-13), 41.9 (C-9), 39.8 (C-10),
38.4 (C-2), 36.9 (C-5), 36.4 (C-4), 36.4 (C-12), 34.5 (C-15),
32.9 (C-23), 32.3 (C-25), 22.9 (C-24), 22.2 (C-18), 21.6
(C6H4–CH3), 19.0 (C-11), 16.3 (C-27), 12.2 (C-19), 11.7
(C-21). HRMS (FAB) m/z calcd for C34H47O6S [M + H]+,
583.3093, found: 583.3079.
5.1.3. Semi-quantitative RT-PCR analysis for
apoptosis-related signals
5.1.3.1. mRNAs. Hep G2 cells (1 × 106) were seeded in 10 cm
dishes. Each dish was cultured with 12 ml of DMEM con-
taining 10% FBS and a penicillin–streptomycin mixture at
37 °C in a humidified atmosphere containing 5% CO2 in air.
After 24 h, the medium was replaced with test medium
containing vitamin D-type derivative 12, spirostanol 26 and
furostanol 36. Hep G2 cells were treated with these test drugs
at their IC50 values for 48 h. All experiments were performed
separately at least three times. Samples of mRNA were
obtained from each culture at 48 h using a total RNA extrac-
tion kit (Rneasy Mini Kit) (Qiagen GmbH, Hilden, Ger-
many). The RT-PCR primers were 5′-GCA CTG GTG TTT
TGT TGT GG-3′ and 5′-GTG GTT TCA AGG CCA GAT
GT-3′ for p53 primers (304 bp), 5′-AAG CTG AGC GAG
TGT CTC AAG CGC-3′ and 5′-ACC ACT GTG ACC TGC
TCC AGA AG-3′ for Bax primers (424 bp), 5′-GAC ACC
ACT GGA GGG TGA CT-3′ and 5′-CAG GTC CAC ATG
GTC TTC CT-3′ for p21 primers (172 bp), 5′-AGA TGT CCA
GCCAGC TGCACC TGA C-3′ and 5′-AGC CTC CGT TAT
CCT GGA TCCA-3′ for Bcl-2 primers (242 bp), and 5′-CAA
TAT GAT TCC ACC CAT GGC AAA TTC CAT GGC AC-3′
and 5′-TGA AGT CAG AGG AGA CCA CCT GGT GCT
5.1.1.17. (25R)-Furost-1␣,3b-dihydroxy-26-thiocyano-
6,8(14),20(22)-triene (36) from 38. The general procedure
for iodination was employed for 38 (18 mg, 0.03 mmol) to
give crude 39 (15 mg), then the same reaction of 39 with
KSCN (6 mg, 0.06 mmol) as described for 35 gave com-
1
pound 36 (11 mg, 75.8%). The FABMS spectrum and H-
and 13C-NMR spectra of 36 were agreed with those of the
compound obtained from 31.
5.1.2. Cytotoxic activities
5.1.2.1. Cell lines and culture. The human colorectal carci-
noma cell line (HCT 116, ATCC No. CCL-247) and human
hepatoma cell line (Hep G2 No. RCB0459) were purchased
from Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan) and
RIKEN Cell Bank (Tsukuba, Japan), respectively. Dulbec-

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